The present invention relates to a fusion protein of a component of an internalizing receptor system and a moiety that binds to a specific cellular surface marker on a cell, to a conjugate of a toxin and a ligand for the internalizing receptor system, and to a method of tumor therapy using the conjugate and internalizing receptor system.
There is now a fairly large and growing body of experience in the use of monoclonal antibodies (mAbs) for the therapy of lymphoma. Several studies targeting different B-cell restricted CD (clusters of differentiation) antigens have shown promising results. These studies have used radiolabeled mAbs and, to a lesser extent, mAb-toxin conjugates, and have targeted CDs19-22, CD37, and HLA-DR.
MAbs used in lymphoma therapy differ in their ability to bind cognate antigen and to become internalized. For example, CD22 exhibits efficient internalization as well as reexpression of antigen after internalization. It suffers, however, from relatively low expression levels on most B-cell malignancies, and is not widely expressed, e.g., it is expressed on only 30-50% of cases of B-cell lymphocytic leukemia (B-CLL).
The present inventor has studied an anti-CD22 mAb, LL2. Preliminary studies using LL2 labeled with 131I for both therapy and imaging of NHL have produced response rates of 30-90+%, with varying percentages of complete responses and differences in durability of response. Higher response rates and longer disease-free survival have been associated with higher total doses of antibody and of radioactivity, which usually have required autologous bone marrow or peripheral stem cell rescue. While the results are encouraging, it is desired to increase therapeutic efficacy and decrease toxicity, particularly myelotoxicity.
The CD20 antigen, in contrast to the CD22 antigen, is a quite highly expressed B-cell restricted antigen that is expressed on a wide range of B-cell malignancies, ranging from acute lymphocytic leukemia (ALL) to the more differentiated B-Cell (B-CLL) and non-Hodgkin""s lymphoma (NHL), and even to hairy cell leukemia (HCL). It generally is expressed on cells in the vast majority of cases of these malignancies at a high antigen density. A major disadvantage of CD20 is that it is a slowly internalizing antigen. For RAIT directed against CD20 this feature may not be a problem, but it militates significantly against the use of CD20 for toxin-based therapy.
A further problem of CD20 is the fact that B-cell malignancies exhibit a more rapid dissociation of bound anti-CD20 mAbs from the surface as compared to nonlymphoma tumor cells. This suggests that a therapy that uses bonding to a B-cell restricted antigen, particularly those characterized by slow internalization, would not be successful.
A variety of mAb-toxin constructs have been tested in both in vitro experiments and human trials. These studies have demonstrated potent and specific effects of these reagents. Most of the toxin molecules that have been used derive from either plant or bacterial sources and hence produce allergenic sensitization in patients. This severely limits the duration of therapy.
While major progress has been made in the therapy of B-cell malignancies such as NHL and B-CLL, there remain a substantial number of patients with B-cell malignancies who exhibit primary resistance to, or relapse after, optimal chemotherapy. A therapy that is effective over long periods of time in most or all patients with B-cell malignancies is desired.
It is therefore an object of the present invention to provide a more effective and less toxic anti-tumor therapy, particularly a therapy for treatment of B-cell malignancies, such as NHL and B-CLL.
It is another object of the invention to improve the value as antigenic targets of slowly internalizing surface antigens such as the CD20 antigen.
It is a further object of the invention to overcome the tendency of antibodies bound to the surface of lymphoma cells to dissociate rapidly from the surface of the cells.
It is yet another object of the present invention to use B-cell restricted antigens, particularly the CD20 antigen, in anti-tumor therapy.
These and other objects of the invention are achieved by providing a conjugate of toxin or therapeutic radionuclide and IL-15, and a fusion protein comprising a bispecific antibody that has a first specificity for a cell marker specific to a malignant cell and a second specificity for a region of IL-15xcex1, each optionally further comprising a diagnostic radionuclide, which are useful therapeutic reagents for treating leukemias and lymphomas.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
It has been discovered, surprisingly, that the value of surface antigens as antigenic targets can be improved significantly by functionally linking them to a high affinity, internalizing receptor system. The present invention is of particular advantage in the case of surface antigens that do not internalize or that internalize slowly. A preferred example of a high affinity, internalizing receptor system is the IL-15 receptor system. When the IL-15 receptor system is used, it can be employed with all malignant cells that contain the xcex2/xcex3c chains of IL-15 receptor. The presence of xcex2/xcex3c chains of IL-15 on the cells provides the basis for a continuously internalizing receptor system that can be bridged to a surface antigen, particularly a slowly internalizing antigen, by way of a bispecific fusion protein and cognate ligand. The method according to the invention results in increased intracellular delivery into the malignant cell of cytotoxic ligands. It also improves methodologies in which a radionuclide is used as a therapeutic agent, by producing a tighter binding of the radionuclide to the malignant cell and by reducing dissociation of the targeting agent from the cell surface.
In accordance with the invention, malignant cells are pretargeted with a fusion protein. The fusion protein comprises a region of IL-15xcex1, preferably an extracellular domain, and a bispecific antibody or antibody fragment that has a first specificity for a cell marker specific to a malignant cell marker and a second specificity for the region of IL-15xcex1. The fusion protein is positioned on the malignant cells by means of the surface antigen expressed by the malignant cells. In an alternative embodiment, the fusion protein is formed in situ, by first administering the bispecific antibody, and then administering IL-15xcex1 which binds to the bispecific antibody that is already bound to the malignant cells. In either case, addition of an armed ligand comprising IL-15 ligand armed with a toxin or with a radionuclide then results in the formation of a trimeric complex of the xcex2/xcex3c chains of IL-15 receptor, in which the xcex1-chain of IL-15 receptor attached to the surface antigen and IL-15/toxin and/or radionuclide conjugate. Alternatively, both the fusion and the trimeric complex can be formed in situ. This leads to rapid internalization of toxin and/or radionuclide into the malignant cells. While internalization is not necessary for a therapeutic radionuclide to be effective, the trimeric complex provides a tighter binding to the malignant cells, and thus improves these modalities as well.
Receptor complexes for both IL-2 and IL-15 have three primary chains. The xcex2 and xcex3c chains are common to the two receptors, and there are individual, private alpha chains, IL-2Rxcex1 and IL-15Rxcex1. The IL-2/IL-2 receptor system consists of at least three subunits, IL-2Rxcex1, IL-2Rxcex2 and IL-2Rxcex3c. This multi-subunit receptor is capable of binding ligand with high affinity and the ligand/receptor complex is rapidly internalized (t1/2≈15 min). IL-2Rxcex1 when expressed in the absence of the other two chains internalizes slowly, and is unable to transduce a signal when expressed by itself. When IL-2Rxcex1 is juxtaposed to the other subunits by the presence of ligand the entire ligand/xcex1xcex2xcex3 complex internalizes at the rapid rate intrinsic to the IL-2Rxcex2/xcex3c dimer. IL-2Rxcex1 thus raises the affinity of the xcex2/xcex3c complex from Ka≈109 to ≈1011 Mxe2x88x921.
IL-15Rxcex1 is structurally similar to IL-2Rxcex1, and is of similar size. As compared to IL-2Rxcex1, IL-15Rxcex1 has an affinity for its cognate ligand (Kaxe2x89xa71010 Mxe2x88x921) that is at least two orders of magnitude greater than that of IL-2Rxcex1 for its ligand. IL-15Rxcex1, like IL-2Rxcex1, has a short intracytoplasmic domain and is unable to transduce a signal when expressed by itself. Thus, the IL-15/IL-15R system operates in a similar fashion to the IL-2/IL-2R system and will internalize all three of its receptor components.
The antigen to which the fusion protein containing the IL-15Rxcex1 is anchored is one that is specific to the malignant cell type. In a preferred embodiment, the antigen is a high-density B-cell restricted antigen. As shown herein, there is expression in malignant B-cells of the xcex2 and xcex3 chains of IL-15 receptor, and little or no expression of the receptor. The presence of xcex2/xcex3c chains of IL-15 receptor on malignant B-cells forms the basis for a continuously internalizing receptor system that can be used in conjunction with B-cell restricted antigens specifically to introduce toxin, and optionally radionuclides, into malignant B-cells. This system can be self-amplifying in that internalized receptors can be either recycled or resynthesized and expressed.
For treatment of NHL, B-CLL, HCL and ALL, the high-density CD20 antigen is a particularly suitable surface antigen. For ALL or multiple myeloma, CD38 is suitable, while for acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML), the CD15 antigen can be used. In addition a variety of solid tumor surface antigens have been described, and any of these can be used in accordance with the present invention.
A bispecific antibody-based molecule, preferably a Mab, is used as the vehicle to position the xcex1-chain of the IL-15 receptor on the surface of the targeted cells. Positioning large amounts of IL-15Rxcex1 on cells that already express the xcex2/xcex3c chains of IL-15 receptor will, after addition of armed IL-15 ligand, induce internalization of this ligand/receptor complex by interaction with the xcex2 and xcex3c chains of IL-15 receptor already present on the cells.
Murine Mab frequently induce human-anti-mouse antibodies (HAMA). When such Mab are used in the present invention, this problem of immunogenicity is minimized by genetically engineering the murine Mab using either chimerization or humanization. Both strategies involve the replacement of some part of the murine sequences with human immunoglobulin sequences. In the chimeric approach the constant regions are replaced with corresponding human sequences. With humanization there is additional replacement of framework sequences within the variable regions of the heavy and light chain genes. Both of these approaches have, in fact, resulted in Mabs with lower immunogenicity. For example, the LL2 antibody has been humanized with retention of its native ability to bind antigen and become internalized, as disclosed in copending application Ser. No. 08/289,576, which is incorporated herein by reference in its entirety.
Mab engineering techniques have been used to produce another class of antibody molecule, namely the single chain antibody, scFv. This molecule is produced by cloning the VH and VL segments from the Mab of interest and splicing them together with a short linker region interposed between them. These molecules, after proper design and renaturation, retain the antigen binding activity of the parent Mab and can be expressed at high levels in E. coli-based expression systems. These constructs then can provide a platform for the engineering of bifunctional single chain molecules that can link a second antigenic target to the first to retarget effector cells or molecules.
The invention utilizes pretargeting of the antigenic target with the fusion protein comprising the Mab or Fc fragments connected to a region of IL-15Rxcex1. In this approach, enhanced tumor/normal tissue ratios of the Mab or Fc fragment are achieved by giving the nontoxic first reagent that has reactivity to the antigenic target. This is followed by a tumor targeting/washout interval that allows for uptake by tumor masses of this first agent and its clearance from normal tissues, after which the toxic conjugate is given.
Prior to the pretargeting with the fusion protein containing the region of IL-15xcex1, the cells may be pretargeted with streptavidin-conjugated antibodies or biotinylated antibodies in conjunction with avidin and biotin. For example, biotinylated anti-CD20 antibodies can be administered, followed by administration of avidin to provide additional binding sites. Subsequently administered biotinylated IL-15Rxcex1 then attaches to the avidin sites.
Both two-step and three-step methods that utilize avidin-biotin chemistry can be employed. These generally involve, depending on the specific protocol, the administration of either avidin- or biotin-conjugated-mAb. This is followed, after an interval of 1-3 days, by the injection of biotin or avidin that is labeled with either a gamma-emitting radionuclide for imaging or by a beta or alpha emitter for therapy. The three-step method interposes a clearing step between the pretargeting and targeting steps. This step promotes the clearance of circulating, residual pretargeting agent, thereby reducing this pool and subsequent access to it by the targeting agent. In this system avidin is given to promote clearance since its elimination kinetics show a very rapid initial phase with a t1/2=1 min which accounts for the majority of the total and a second phase with a t1/2≈30 min.
The methods that use biotin and avidin lead to an increased number of sites for binding of active conjugate, but these improvements are mitigated by the fact that both avidin and the alternate protein, streptavidin, are immunogenic. Somewhat less than 30% of patients develop antibodies to avidin and a full 70% of patients develop antibodies to streptavidin. Accordingly, it is less preferable to use a pretargeting with biotin/avidin.
In a preferred embodiment according to the invention, therefore, a two-step procedure is used in which only the fusion protein containing the region of IL-15xcex1 fusion protein, in its optimal humanized form, is used to pretarget the malignant cells. The fusion protein according to the present invention has low immunogenic potential. The pretargeting agent bears human IL-15Rxcex1, which has a high affinity for ligand. This fusion protein, owing to its relatively low molecular weight (70 kDa vs. 150 kDa for intact IgG), has the potential for greater penetration into the interior of tumors.
The second step reagent, the IL-15 construct, likewise has low immunogenic potential and a low molecular weight (rIL-15 from E. coli has a MW of 13 kDa) to aid in both tumor penetration and clearance from non-tumor sites. It is administered after the pretargeting, IL-15Rxcex1 fusion protein has localized on the malignant cells and substantially cleared from the circulation. This system could also be adapted to include a third step if this were necessary, i.e., an intervening preclearance. This can be done by galactosylating the IL-15 ligand. For higher galactose substitution IL-15 can be crosslinked covalently to asialofetuin.
The IL-15 ligand can be armed with a radionuclide or a toxin. The radionuclide can be either a diagnostic or therapeutic radionuclide. In a preferred embodiment, the IL-15 ligand is used to administer both radionuclide and toxin. The same IL-15 ligand can be armed with both radionuclide and toxin, or separate IL-15 ligands can be armed with radionuclide and toxin. Where separate IL-15 ligands armed with radionuclide and toxin are used, these may be administered together or sequentially.
When the IL-15 ligand is armed with a toxin, a preferred toxin is a ribonuclease, such as onconase. Onconase is a non-mammalian RNAse purified from Rana pipiens oocytes. It has been shown in clinical trials to have anti-tumor activity against human pancreatic cancer, but has been found to have minimal anti-tumor activity against B-cell malignancies such as B-cell lymphocytic leukemia.
The fusion protein and armed ligand conjugate are administered in a composition with a pharmaceutically acceptable carrier. In this regard, a pharmaceutically acceptable carrier is a material that can be used as a vehicle for administering the fusion protein or armed ligand because the material is inert or otherwise medically acceptable, as well as compatible with the fusion protein or armed ligand.
Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For example, since the xcex2/xcex3c chains of IL-15 receptor are the same as the xcex2/xcex3c chains of IL-2 receptor, the fusion protein can be used to introduce IL-2Rxcex1 onto the malignant cells, followed by administration of an RNase-IL-2 conjugate. Moreover, fusion proteins of either IL-15Rxcex1 or IL-2Rxcex1 can be made in which the fusion partner is an antibody other than an anti-CD20 antibody. This enables pretargeting of any tumor that carries a specific marker. Many antibodies and antibody fragments which specifically bind markers produced by or associated with tumors or infectious lesions have been disclosed, inter alia, in Hansen et al. U.S. Pat. No. 3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459 and 4,460,561. Where the tumor additionally contains the xcex2/xcex3c chains of IL-15/IL-2 receptors, the fusion protein will be rapidly internalized.
The following examples are illustrative of the present invention, but are not to be construed as limiting.